Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

• Published in: Archives of biochemistry and biophysics 2007-09, Vol.465 (2), p.347-358
• Main Authors: Wang, Shuli, Lee, Douglas P., Gong, Nan, Schwerbrock, Nicole M.J., Mashek, Douglas G., Gonzalez-Baró, Maria R., Stapleton, Cliona, Li, Lei O., Lewin, Tal M., Coleman, Rosalind A.
• Format: Article
• Language: English
• Subjects: Animals; Chlorocebus aethiops; Cloning, Molecular - methods; COS Cells; Enzyme Activation; Glycerol-3-Phosphate O-Acyltransferase - chemistry; Glycerol-3-Phosphate O-Acyltransferase - immunology; Glycerol-3-Phosphate O-Acyltransferase - metabolism; Glycerolipid; Gpam; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria - enzymology; Organ Specificity; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Steatosis; Testis; Testis - enzymology; Tissue Distribution; Triacylglycerol
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2007.06.033

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25 μCi) [1- 14C]oleate for 6 h increased incorporation of [ 14C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.